苯通過激活線粒體凋亡通路對小鼠骨髓細胞凋亡的誘導作用及其機制

于光艷,宋祥福,趙淑華,劉曉梅,孫志偉,2

(1.吉林大學公共衛(wèi)生學院勞動衛(wèi)生與環(huán)境衛(wèi)生教研室,吉林長春 130021;2.首都醫(yī)科大學公共衛(wèi)生學院衛(wèi)生毒理學教研室,北京 100069)

苯通過激活線粒體凋亡通路對小鼠骨髓細胞凋亡的誘導作用及其機制

于光艷1,宋祥福1,趙淑華1,劉曉梅1,孫志偉1,2

(1.吉林大學公共衛(wèi)生學院勞動衛(wèi)生與環(huán)境衛(wèi)生教研室,吉林長春 130021;2.首都醫(yī)科大學公共衛(wèi)生學院衛(wèi)生毒理學教研室,北京 100069)

目的:建立小鼠吸入氣態(tài)苯染毒模型,探討苯對骨髓細胞凋亡的誘導作用和機制,為苯骨髓毒作用機制的研究提供實驗依據(jù)。方法:將24只雄性小鼠隨機分為對照組和低、中、高劑量苯染毒組(染毒劑量分別為400、800和1 600 mg·m－3),每組6只。各劑量苯染毒組小鼠進行靜式染毒,每天2 h,連續(xù)染毒15 d后將各組小鼠全部處死,HE染色后光鏡下觀察各組小鼠骨髓細胞的病理學改變,流式細胞儀測定各組小鼠骨髓細胞凋亡率及線粒體膜電位,免疫組織化學法測定各組小鼠線粒體凋亡途徑中相關基因蛋白的表達。結果:低、中和高劑量苯染毒組小鼠骨髓邊緣及中央處細胞數(shù)明顯減少,高劑量苯染毒組小鼠骨髓還伴有大量的血竇擴張。中和高劑量苯染毒組細胞凋亡率明顯高于對照組,差異有統(tǒng)計學意義(Ρ＜0.01),且高劑量苯染毒組與低和中劑量苯染毒組比較差異也有統(tǒng)計學意義(Ρ＜0.05)。低、中和高劑量苯染毒組小鼠骨髓細胞線粒體膜電位隨著染毒劑量的增加明顯降低,中和高劑量苯染毒組與對照組比較差異有統(tǒng)計學意義(Ρ＜0.05或P＜0.01)。不同劑量苯染毒組Bax、CytC及中、高劑量苯染毒組Caspases-9、Caspases-3陽性細胞數(shù)隨染毒劑量的增加顯著升高,與對照組比較差異均有統(tǒng)計學意義(Ρ＜0.05);而各劑量苯染毒組Bcl-2陽性細胞數(shù)則明顯低于對照組(Ρ＜0.05),且中和高劑量苯染毒組與低劑量苯染毒組比較差異也有統(tǒng)計學意義(Ρ＜0.05)。結論:一定劑量的苯可以誘導小鼠骨髓細胞發(fā)生凋亡,促進線粒體凋亡相關基因蛋白的表達。通過線粒體凋亡通路誘導的細胞凋亡可能是苯骨髓毒性的重要機制之一。

苯;細胞凋亡;線粒體膜電位;凋亡基因蛋白

苯是一種在工農業(yè)生產(chǎn)及日常生活中應用很廣泛的有機溶劑,普遍存在于環(huán)境中。流行病學和動物實驗研究[1-5]表明:長期接觸苯會引起血液系統(tǒng)方面的改變,導致白細胞減少和骨髓抑制,發(fā)生白血病和惡性淋巴瘤的危險性升高。盡管國內外專家學者對苯的毒作用機制已經(jīng)進行了多方面的研究和探討,但其致血液毒性的機制迄今尚未完全闡明。近年來研究[6-9]顯示:苯可以誘導造血細胞發(fā)生凋亡,且凋亡相關基因的差異表達與苯血液毒性的發(fā)生、發(fā)展有密切關聯(lián)。如果對苯誘導的細胞凋亡相關基因及調控凋亡的信號轉導通路進行研究,可為闡明苯中毒的發(fā)病機制提供重要依據(jù)[5]。本研究通過建立小鼠吸入氣態(tài)苯染毒模型,采用細胞生物學、免疫學等實驗方法,探討細胞凋亡相關信號轉導通路在苯誘導的小鼠骨髓細胞凋亡中的作用,為闡明苯中毒的發(fā)病機制和預防白血病的發(fā)生及降低其發(fā)病率提供實驗依據(jù)。

1 材料與方法

1.1 實驗動物與分組24只普通級封閉群昆明系雄性小鼠由吉林大學實驗動物部提供,合格證號: 10-1023,體質量(13±1)g。按照隨機分組原則,參照小鼠吸入苯的半數(shù)致死濃度(LC50) [(45 000±935)mg·m－3·2 h－1],分為對照組和低、中、高劑量苯染毒組(染毒劑量為400、800 和1 600 mg·m－3),進行靜式染毒,每天2 h。連續(xù)染毒15 d后將小鼠全部處死,取骨髓,測定相關指標。

1.2 主要試劑與儀器苯(分析純,天津市致遠化學試劑有限公司),Bax、Bcl-2、CytC、Caspase-3 和Caspase-9多克隆抗體(美國Santa Cruz公司),二氨基聯(lián)苯胺顯色試劑盒(北京中杉金橋生物技術有限公司),SP超敏試劑盒(福州邁新生物技術有限公司),碘化丙啶(PI)、RNA酶和Rhodamine123(美國Sigma公司)。56L靜式染毒柜(吉林大學勞動衛(wèi)生與環(huán)境衛(wèi)生教研室提供), FACScan流式細胞儀(美國Becton Dickinson公司),光學顯微鏡(日本Olympus公司)。

1.3骨髓病理切片制備小鼠眼眶取血處死后,取胸骨,放入10%甲醛溶液中固定,乙醇梯度脫水,常規(guī)石蠟包埋,間斷均勻切片5～10張,切片厚度5μm,HE染色,光鏡下觀察骨髓細胞形態(tài)學變化。

1.4 骨髓線粒體膜電位測定采用流式細胞術(FCM)檢測[10]。將小鼠頸椎脫臼處死,取出股骨,剔除肌肉和結締組織,剪開股骨兩端,用1 m L注射器以0.01 mol·L－1PBS沖出骨髓細胞,濾網(wǎng)過濾(200目尼龍網(wǎng)),制成單個骨髓細胞懸液。加等體積的20 mg·L－1Rhodamine123,至終體積約10 mg·L－1,37℃避光孵育30 min,以PBS清洗脫色,1 500 r·min－1離心10 min,重復1次,用微量0.01 mol·L－1PBS(與沉淀等體積)混勻細胞,采用流式細胞儀(激發(fā)光波長488 nm)檢測,每個樣品收集1×104個細胞,測定細胞相對熒光強度,以U值表示,并以實驗組U值/對照組U值×100%表示細胞線粒體膜電位變化。

1.5 骨髓細胞凋亡檢測采用PI標記,FCM檢測細胞凋亡率[11]。將小鼠頸椎脫臼處死,取出股骨,剔除肌肉和結締組織,剪開股骨兩端,用1 m L注射器以0.01 mol·L－1PBS沖出骨髓細胞,濾網(wǎng)過濾(200目尼龍網(wǎng)),制成單個骨髓細胞懸液。用0.01 mol·L－1PBS洗2次,加100μL RNase(10 mg·L－1)和100μL PI(5 mg·L－1), 4℃避光孵育30 min,用流式細胞儀收集1× 104個細胞,采用Cellquest軟件分析結果,結果以凋亡細胞的百分率表示。

1.6 CytC、Bcl-2、Bax、Caspases-3和Caspases-9蛋白表達的檢測將小鼠頸椎脫臼處死,取胸骨,常規(guī)涂片,自然晾干后,10%甲醛緩沖液固定15 min,風干后放于－20℃冰箱保存。采用免疫組織化學技術[11]進行檢測。PBS洗3次,每次5 min。滴加1∶10稀釋的正常血清,置濕盒內10 min后,每張玻片加1∶50稀釋的第一抗體, 4℃過夜。PBS洗3次,每次5 min,滴加生物素標記的第二抗體,置濕盒內30 min。PBS洗3次,每次5 min,滴加1∶20稀釋的第三抗體(卵白素-生物素過氧化酶復合物),置濕盒內1 h。PBS洗3次,每次5 min,滴加新鮮配制的酶作用底物(DAB),顯色,顯微鏡下觀察染色情況,當目的蛋白出現(xiàn)黃色,立即用自來水沖洗終止顯色反應。蘇木精復染,水洗。置入70%HCl-乙醇中15 s,水洗。弱氨水返藍15 s,顯微鏡下觀察,當胞核呈現(xiàn)藍染,立即水洗。乙醇梯度脫水,二甲苯透明,中性樹膠封片。陽性細胞為胞漿(或胞膜、胞核)呈棕黃色染色。顯微鏡下每張切片隨機觀察5個視野,每個視野觀察100個細胞,陽性細胞在500個細胞中所占的比例為抗體陽性表達率。

1.7 統(tǒng)計學分析采用SPSS 14.0統(tǒng)計軟件對數(shù)據(jù)進行統(tǒng)計處理。小鼠骨髓細胞凋亡率、線粒體膜電位和Bax、Caspases-9、Caspases-3、CytC、Bcl-2蛋白表達水平以±s表示,組間比較采用方差分析和q檢驗。

2 結果

2.1 小鼠骨髓細胞病理學低、中和高劑量苯染毒組骨髓邊緣及中央處細胞數(shù)明顯減少,高劑量苯染毒組還伴有大量的血竇擴張。見圖1(插頁二)。

2.2 各組小鼠骨髓細胞凋亡率及線粒體膜電位中和高劑量苯染毒組小鼠骨髓細胞凋亡率明顯高于對照組,差異有統(tǒng)計學意義(Ρ＜0.01),且高劑量苯染毒組與低、中劑量苯染毒組比較差異也有統(tǒng)計學意義(Ρ＜0.05);各劑量苯染毒組小鼠骨髓細胞線粒體膜電位隨著染毒劑量的增加明顯降低,中和高劑量苯染毒組與對照組比較差異有統(tǒng)計學意義(Ρ＜0.05或P＜0.01)。見表1。

表1 各組小鼠骨髓細胞凋亡率及線粒體膜電位Tab.1 Apoptotic rates of bone marrow cells and mitochondrial membrane potential(MMP)of mice in various groups(n＝4,±s,η/%)

表1 各組小鼠骨髓細胞凋亡率及線粒體膜電位Tab.1 Apoptotic rates of bone marrow cells and mitochondrial membrane potential(MMP)of mice in various groups(n＝4,±s,η/%)

?P＜0.05,??P＜0.01 compared with control group;△P＜0.05 compared with low dose of benzene group;＃P＜0.01 compared with middle dose of bezene group.

Group Apoptotic rate MMP Control 4.40±0.60 23.27±3.01 Benzene Low dose 6.53±1.64 19.48±3.34 Middle dose 11.65±2.35??16.30±1.72?High dose 19.73±2.13??△＃15.64±0.27??

2.3 各組小鼠骨髓細胞中凋亡相關蛋白的表達不同劑量苯染毒組小鼠骨髓細胞中Bax、CytC及中、高劑量苯染毒組小鼠骨髓細胞中Caspases-9 和Caspases-3陽性細胞數(shù)隨染毒劑量的增加顯著升高,與對照組比較差異均有統(tǒng)計學意義(Ρ＜0.05或P＜0.01),中和高劑量苯染毒組與低劑量苯染毒組比較差異有統(tǒng)計學意義(Ρ＜0.05或P＜0.01);而各劑量苯染毒組小鼠骨髓細胞中Bcl-2陽性細胞數(shù)則明顯低于對照組(Ρ＜0.01),且中和高劑量組與低劑量組比較差異有統(tǒng)計學意義(Ρ＜0.05)。見表2。

3 討論

細胞凋亡即細胞程序性死亡,可以由多個基因參與調控。細胞凋亡調節(jié)紊亂可引起細胞增生、分化或異常,從而導致疾病或腫瘤的發(fā)生。有研究[12-13]顯示:細胞凋亡的發(fā)生與多通路的信號轉導及Caspase級聯(lián)反應有關。當細胞受到外界刺激后,可通過線粒體膜通道孔(MPTP)的開放,使線粒體膜電位下降,誘導Cyt C等細胞凋亡相關因子的釋放,激活Caspase-9,繼而活化Caspase-3,啟動Caspase級聯(lián)反應,引起細胞凋亡[14-15]。在這一過程中,Bcl-2家族蛋白對于MPTP的開放和關閉起著關鍵的調節(jié)作用。促凋亡類蛋白Bax等可以通過與線粒體內膜上的腺苷酸轉運蛋白(ANT)或外膜上的電壓依賴性陰離子通道(VDAC)結合介導MPTP的開放,促進Cyt C的釋放;而抗凋亡類蛋白Bcl-2等則通過與Bax競爭性地與ANT結合或直接阻止Bax與ANT、VDAC的結合發(fā)揮其抗凋亡效應[16-17]。

表2 各組小鼠骨髓細胞中Bax、Caspases-9、Caspases-3、CytC和Bcl-2陽性細胞數(shù)Tab.2 Number of Bax,Caspases-9,Caspases-3,CytC and Bcl-2 positive cells in bone marrow cells of mice in various groups (n＝5,±s,η/%)

表2 各組小鼠骨髓細胞中Bax、Caspases-9、Caspases-3、CytC和Bcl-2陽性細胞數(shù)Tab.2 Number of Bax,Caspases-9,Caspases-3,CytC and Bcl-2 positive cells in bone marrow cells of mice in various groups (n＝5,±s,η/%)

?P＜0.05,??P＜0.01 compared with control group;△P＜0.05,△△P＜0.01 compared with low dose of benzene group.

Group Bax Caspases-9 Caspases-3 CytC Bcl-2 Control 13.00±1.41 9.80±1.92 11.20±1.48 10.20±1.92 54.60±1.34 Benzene Low dose 16.80±1.48?11.80±1.30 14.00±2.24 14.40±1.67?42.00±3.39??Middle dose 38.80±4.09?△19.40±3.78?△30.80±4.87??△△29.00±1.87??△△35.80±0.84??△High dose 43.00±3.39?△24.00±2.45??△△39.40±6.19??△△32.80±4.97??△△31.60±3.21??△

本研究結果表明:一定劑量的苯可以降低線粒體膜電位,誘導骨髓細胞發(fā)生凋亡。苯作為化學物質,對機體的刺激被細胞接受后,細胞可以將這些信號進行整合,通過細胞凋亡信號與特異性受體結合成復合物,激活Caspase,促使其對特異性底物進行降解,導致凋亡抑制失活,引起細胞凋亡。本研究采用免疫細胞化學方法檢測小鼠骨髓細胞中凋亡相關蛋白CytC、Bax、Bcl-2、Caspase-9和Caspase-3蛋白表達的結果顯示:一定劑量的苯可以誘導骨髓細胞凋亡相關蛋白的表達,使Cyt C、Bax、Caspase-9和Caspase-3陽性細胞數(shù)升高,而Bcl-2陽性細胞數(shù)降低。本研究結果表明線粒體凋亡通路參與了苯誘導的小鼠骨髓細胞凋亡,這可能是苯引起骨髓毒性的主要機制之一。

[1]Robert Schnatter A,Kerzic PJ,Zhou Y,et al.Peripheral blood effects in benzene-exposed workers[J].Chem Biol Interact,2010,184(1/2):174-181.

[2]Sorahan T.Occupational benzene exposure and lymphoma risks[J].Environ Health Perspect,2011,119(11):A468-469.

[3]Wong O,Harris F,Armstrong TW,et al.A hospital-based case-control study of acute myeloid leukemia in Shanghai: analysis of environmental and occupational risk factors by subtypes of the WHO classification[J].Chem Biol Interact, 2010,184(1/2):112-128.

[4]Vlaanderen J,Lan Q,Kromhout H,et al.Occupational benzene exposure and the risk of lymphoma subtypes:a metaanalysis of cohort studies incorporating three study quality dimensions[J].Environ Health Perspect,2011,119(2): 159-167.

[5]劉 揚,賀 今,劉 軍.苯中毒細胞凋亡相關基因研究進展[J].職業(yè)與健康,2011,27(13):1542-1543.

[6]Zhang L,Mchale CM,Rothman N,et al.Systems biology of human benzene exposure[J].Chem Biol Interact,2010, 184(1/2):86-93.

[7]Peng D,Jiaxing W,Chunhui H,et al.Study on the cytogenetic changes induced by benzene and hydroquinone in human lymphocytes[J].Hum Exp Toxicol,2012, 31(4):322-335.

[8]Sappayatosok K,Maneerat Y,Swasdison S,et al.Expression of proinflammatory protein,iNOS,VEGF and COX2 in oral squamous cell carcinoma(OSCC),relationship with angiogenesis and their clinicopathological correlation[J].Med Oral Patol Oral Cir Bucal,2009,14(7):319-324.

[9]劉 軍,王秀英.苯中毒患者骨髓單個核細胞凋亡調節(jié)基因的表達及其意義[J].新醫(yī)學,2010,41(11):745-746.

[10]王志成,李艷博,郭 偉,等.低劑量電離輻射對小鼠睪丸生精細胞中活性氧活性和線粒體膜電位的影響[J].吉林大學學報:醫(yī)學版,2007,33(5):786-789.

[11]龔守良.實用醫(yī)學實驗技術[M].長春:吉林科學技術出版社,1991:290.

[12]Fan XX,Li N,Wu JL,et al.Celastrol induces apoptosis in gefitinib-resistant non-small cell lung cancer cells via caspasesdependent pathways and Hsp90 client protein degradation[J].Molecules,2014,19(3):3508-3522.

[13]岳原亦,張 揚,張一奇.Caspase家族與細胞凋亡[J].中國醫(yī)療前沿,2011,6(6):25-26.

[14]周藝群,谷志遠.線粒體與細胞凋亡[J].解剖科學進展, 2006,12(1):60-62.

[15]Ola MS,Nawaz M,Ahsan H.Role of Bcl-2 family proteins and caspases in the regulation of apoptosis[J].Mol Cell Biochem,2011,351(1/2):41-58.

[16]郎海濱.Bcl-2家族蛋白與線粒體凋亡路徑研究進展[J].國外醫(yī)學:衛(wèi)生學分冊,2004,31(2):88-91.

[17]Xu JY,Zhou M,Ouyang J,et al.Gambogic acid induces mitochondria-dependent apoptosis by modulation of Bcl-2 and Bax in mantle cell lymphoma JeKo-1 cells[J].Chin J Cancer Res,2013,25(2):183-191.

Induction effect of benzene on apoptosis of mouse bone marrow cells through mitochondrial-dependent apoptosis pathway and its mechanism

YU Guang-yan1,SONG Xiang-fu1,ZHAO Shu-hua1,LIU Xiao-mei1,SUN Zhi-wei1,2
(1.Department of Occupational and Environmental Health,School of Public Health,Jilin University, Changchun 130021,China;2.Department of Hygienic Toxicology,School of Public Health,Capital Medical University,Beijing 100069,China)

ObjectiveTo establish mouse poisoning model by inhaling benzene,and to investigate the induction effect of benzene on the apoptosis of mouse bone marrow cells and its mechanism,and to provide an experimental basis for study on bone marrow toxicity mechanism.Methods24 male mice were randomly divided into four groups (n＝6).The mice in one group were exposed to ambient air(control group)and the mice in the other three groups were exposed to different doses(400,800,1 600 mg·m－3)of benzene(low,middle and high doses of benzenegroups)for 15 d in the respective inhalation chambers.At the end of the experiment,the mice were killed.The bone marrow of the mice was obtained.The pathological changes of the bone marrow cells of the mice in various groups were observed under light microscope with HE staining.The apoptotic rates and mitochondrial membrane potential(MMP)of the mice in various groups were detected by flow cytometry,and the expressions of mitochondrial-deperdent apoptosis related gene proteins were determined with immunohistochemistry method.ResultsThe number of distal and central cells in different doses of benzene groups were significantly reduced,and accompanied by blood sinus expansion in high dose of benzene group.The apoptotic rates of the cells in middle and high doses of benzene groups were obviously higher than that in control group(Ρ＜0.01),and there were also significant differences between high dose group and low,middle doses of benzene groups(Ρ＜0.05).The MMP was significantly decreased with the increasing of benzene doses,and there were significant differences between middle,high doses of benzene groups and control group(Ρ＜0.05).The number of Bax,CytC positive cells in different doses of benzene groups and the number of Caspase-9,Caspase-3 positive cells in middle and high doses of benzene groups were significantly increased compared with control group(Ρ＜0.05);the number of Bcl-2 positive cells in different doses of benzene groups was decreased(Ρ＜0.05),and number of Bcl-2 positive cells in middle and high doses of benzene groups was decreased compared with low dose of benzene group(P＜0.05).ConclusionBenzene with certain dose can induce the apoptosis of mouse bone marrow cells,and promote the expressions of mitochondrial apoptosis related gene proteins.Benzene-induced apoptosis through mitochondrialdependent apoptosis pathway may be an important mechanism of bone marrow toxicity induced by benzene.

benzene;apoptosis;mitochondrial membrane potential;apoptosis gene protein

R114;R363

A

2013-11-09

國家安監(jiān)總局計劃項目資助課題(08-067);中央高校基本科研業(yè)務費專項基金資助課題(450060481119)

于光艷(1976－),女,吉林省吉林市人,講師,醫(yī)學博士,主要從事工業(yè)及環(huán)境毒理學研究。

孫志偉(Tel:010-83911507,E-mail:zwsun＠hotmail.com)

1671-587Ⅹ(2014)05-0943-04

10.13481/j.1671-587x.20140507